海藻酸钠-多聚赖氨酸-海藻酸钠微囊化胰岛细胞移植治疗糖尿病的实验研究

采用Ｓｕｎ海藻酸钠－多聚赖氨酸－海藻酸钠（ＡＰＡ）微囊制作技术,分别包裹大鼠胰岛和胰岛素分泌细胞系,移植于糖尿病小鼠腹腔。结果表明ＡＰＡ微囊化大鼠胰岛或胰岛素分泌细胞移植,均可使糖尿病小鼠血糖降低至接近正常水平达３周至１１０天;移植微囊无明显的组织学反应。证明该ＡＰＡ微囊化胰岛细胞移植具有较好的治疗效果,微囊具有较好的生物相容性和免疫隔离作用。为进一步发展生物型人工胰岛奠定了基础。     

海藻酸钠—多聚赖氨酸—海藻酸钠微囊化胰岛细胞移植治疗糖尿 …

采用Ｓｕｎ海藻酸钠－多聚赖氨酸－海灌酸钠（ＡＰＡ）微囊制作技术，分别包裹大鼠胰岛素分泌细胞系，移植于糖尿病小鼠腹腔。结果表明ＡＰＡ微囊化大鼠胰岛或胰岛素分泌细胞，均可使糖尿病小鼠血糖降低至拉近正常水平达３周至１１０天，移植微囊无明显的组织学反应。证明该ＡＰＡ微囊化胰岛细胞移植具有较好的治疗效果，微囊具有较好的生物相容性和免疫隔离作用。进一步发展生物型人工胰尊定了基础。     

大鼠胰岛分离与纯化的实验研究

为探讨胰岛移植物的制备方法，采用胰管注射胶原酶、胰腺静止消化方法，分离成年大鼠胰岛，葡聚糖离心纯化。纯化后胰岛收获量为６１０～８２０个／胰腺，纯度达９２％；胰岛形态结构完整，内分泌细胞超微结构保持良好；对葡萄糖刺激反应胰岛素释放量是基础分泌水平的８倍；异种移植可逆转实验性糖尿病小鼠的高血糖达１周。结果表明：胰管注射胶原酶胰腺静止消化可获得高产量、高纯度、功能良好的胰岛。为临床胰岛移植开辟了新的途径。     

糖尿病犬经肝动脉肝内移植微囊化猪胰岛细胞的安全性和有效性

目的评估移植于I型糖尿病犬肝脏中的微囊化新生猪胰岛细胞(NPI)的生物相容性、免疫学特性及生理学特性。方法I型糖尿病犬分为A、B两组,每组15只,A组每只犬分别经肝动脉灌注微囊化新生猪胰岛细胞40～60万个,B组每只犬分别经肝动脉灌注未微囊化新生猪胰岛细胞40～60万个,两组动物移植后均不使用免疫抑制治疗。移植前后分别测量移植受体的肝脏功能及淋巴细胞CD4 /CD8 比值。移植6个月后所有移植受体的肝脏均进行病理学检查。结果移植后A组外源性胰岛素用量从移植前的22 u逐渐降至5 u,B组所需外源性胰岛素从移植前的24 u下降至10 u。移植后2～3周B组胰岛素用量恢复到移植前的水平,而A组的部分动物的胰岛素剂量继续减至8 u。B组受体移植后血的CD4 较移植前升高,而A组的CD4 和CD8 细胞移植后无明显变化。移植后所有受体的肝功能及组织结构未见异常。结论微囊化的新生猪胰岛细胞在受体犬的肝脏中有很好的生物相容性。微囊可以延长移植物的存活,且异种移植微囊化的新生猪胰岛细胞能够纠正糖尿病犬的高血糖状态。     

APA微囊化胰岛研究进展

微囊化胰岛是从20世纪80年代发展起来的细胞移植治疗糖尿病的重要方法。海藻酸-聚赖氨酸-海藻酸微囊(APA)具有广阔的应用前景。本文综述了近年来微囊化胰岛的研究进展，重点分析了微囊材料的生物相容性，微囊的完整性，微囊的移植部位等影响APA微囊治疗效果的因素，以期为完善微囊化胰岛的治疗效果提供线索。     

微囊技术在生物医学中应用的研究进展

微囊技术（encapsulation）是指用半透膜包被生物活性物质以形成微囊的技术，形成的微囊可以阻遏免疫细胞以及大分子抗体通过半透膜，同时允许氧气、营养物质和一些具有生物活性的小分子物质自由出入，因此，微囊技术是目前细胞治疗、组织和器官替代治疗的主要方法之一。微囊技术的应用可以追溯到1933年，Bisceglie运用复合膜包被肿瘤细胞移植到猪的腹腔，结果发现移植的细胞并没有受到免疫系统的破坏。1964年，Chang再次引用微囊技术进行细胞移植，并首次提出“人工细胞”的概念。随后的20年问，人们不断尝试用这种方法包埋胰岛细胞来控制糖尿病小动物模型的高血糖。1980年，Lim等首次成功地用微囊化胰岛细胞移植纠正糖尿病动物高血糖。1986年，0’Shea等在已有基础上对成囊物质进行了革新，制成了海藻酸钠多聚赖氨酸海藻酸钠（APA）微囊。近几年来，许多实验室探索采用其他方法和材料研制新型微囊，试图改善微囊的一些生物学和化学特性，以使其能够在代谢性疾病的治疗、药物缓释控制、体内和体外细胞培养等领域得到广泛应用。本文仅就微囊技术在生物医学领域中的应用进行综述。     

模拟微重力条件下大鼠胰岛培养及体内移植的实验研究

目的研究经模拟微重力条件培养的胰岛是否能够降低胰岛移植过程中引发的免疫排斥反应，进而延长胰岛的体内存活时间。方法分离新鲜胰岛，应用培养皿对胰岛进行静态培养，同时应用旋转式生物反应器对胰岛进行三维立体培养。应用吖啶橙一碘化丙啶（AO-PI）染色和葡萄糖刺激实验对3种条件下培养得到的胰岛进行生物学活性研究。2000当量胰岛在旋转式生物反应器中培养5d后，将其移植入经链脲佐菌素（STZ）处理的糖尿病模型SD大鼠。肾被膜下作为实验组，以新鲜分离的胰岛和静态培养5d的胰岛移植入经STZ处理的糖尿病模型SD大鼠肾被膜下作为对照组。在不同时间点上，检测各组SD大鼠血糖变化情况；对各移植组中大鼠进行糖耐受实验。切除。肾被膜下的移植物组织作为标本，进行苏木精一伊红（HE）染色及胰岛素组织化学染色检测。结果体外检测结果表明，与新鲜分离的胰岛和静态培养的胰岛相比，微重力条件下培养的胰岛仍保持良好的胰岛素合成及分泌功能。体内实验表明，微重力条件下培养的胰岛体内移植组的血糖保持正常的时间明显高于接受静态培养的胰岛和新鲜分离的胰岛移植组。将微重力培养条件下胰岛移植大鼠后，取。肾被膜下移植区组织标本进行HE染色，未见明显淋巴细胞浸润，胰岛素免疫组织化学均可见阳性细胞。而新分离的胰岛以及静态培养的胰岛移植后，大鼠。肾被膜下移植物组织标本可见淋巴细胞浸润，移植物的厚度明显变小。结论胰岛在微重力条件下培养可降低移植引起的免疫排斥反应，进而延长移植物体内存活时间。     

微囊化周围神经组织腹腔移植的实验研究

目的 观察微囊化周围神经组织移植体内后的生物活性,分析其在体内存活的时效. 方法 采用注射的方法 将微囊化周围神经组织移植到小鼠的腹腔. 结果 微囊化周围神经组织移植6周后,在小鼠的腹腔内保持了原有的形状和结构,囊内的细胞正常生存并保持增殖功能. 结论 微囊化神经组织可在体内保持活性至少6周,完善微囊化技术有望延长其存活时效,提高其促进神经损伤修复的效果.     

第五届全军内分泌学术会议论文综述

会议收到论文216篇，其中内容涉及糖尿病类146篇，眼和甲状腺疾病43篇，其它27篇。现将各部分主要内容综述如下。1糖尿病解放军总医院探讨了弥可保（甲基维生素B12）在形态学和功能学两个方面对糖尿病大白鼠周围神经病变的治疗作用，结果表明，弥可保对周围神经形态改善和感觉神经损害有较好疗效。广州军区总医院报告，应用甲基维生素B12治疗糖尿病性周围神经病变20例，症状改善率达95％，无不良反应。济南军区总医院应用海藻酸钠和氯化钡微囊包被新生猪胰岛行腹腔移植，治疗实验性糖尿病小鼠，可使胰岛素用量减少，血糖下降，2周后停用胰…     

Dextran分离纯化大鼠胰岛的生物学特性

目的探讨一种分离纯化大鼠胰岛的方法,并评价该法分离得到的胰岛的生物学特性。方法常规外科手术分离胰腺。然后将其剪为1mm3左右碎块,置于0·9mg/ml胶原酶V37℃振荡消化10~17min,以含10%胎牛血清的Hank’s液终止消化。60目筛网过滤。Dextran T40非连续密度梯度离心纯化胰岛。纯化后的胰岛经DTZ染色,吖啶橙/碘化丙啶(AO/PI)染色、放射免疫分析检测胰岛素分泌量,纯化后的胰岛进行异种移植,检测降糖效果。结果胰岛分布于11?xtran和1640及11%和22?xtran界面之间,平均每只Wistar大鼠消化后可获得2181±14个胰岛,纯化后回收1826±24个胰岛,胰岛收获量达(85·7±5·9)%,纯度高达95%以上。胰岛对葡萄糖刺激有良好的反应性,纯化后胰岛异种移植,可逆转实验性糖尿病SD大鼠血糖7~10天。结论胶原酶消化,Dextran T40纯化是一种简单、高效的胰岛分离纯化方法,获得的胰岛有良好的生物学特性。     

MR perfusion imaging using encapsulated laser-polarized 3He.

In this work, the use of a new carrier agent for intravascular laser-polarized 3He imaging is reported. Lipid-based helium microbubbles were investigated. Their average diameter of 3 microm, which is smaller than that of the capillaries, makes it possible to conduct in vivo studies. The NMR relaxation parameters T1, T2, and T2* of a microbubble suspension were measured as 90 s, 300 ms, and 4.5 ms, respectively, and in vivo images of encapsulated 3He with signal-to-noise ratios (SNRs) larger than 30 were acquired. Dynamic cardiac images and vascular images of encapsulated 3He were obtained in rats using intravenous injections of microbubble suspensions. Excellent preservation of 3He polarization through the lung capillaries and heart cavities was observed. The first images of 3He microbubble distributions in the lungs were obtained. Additionally, the potential of this technique for lung perfusion assessment was validated through an experimental embolism model with the visualization of perfusion defects.     

T-cell homing to the pancreas in autoimmune mouse models of diabetes: in vivo MR imaging

PURPOSE: To evaluate the efficiency of T-cell labeling with anionic magnetic nanoparticles (AMNPs) and in vivo magnetic resonance (MR) imaging monitoring of T-cell homing to the pancreas. MATERIALS AND METHODS: In vivo MR images of pancreas were obtained with a 7-T MR system in 12 NOD (nonobese diabetic) mice at 11 and 20 days after injection of AMNP-loaded or unloaded T cells. Homing of loaded T cells in pancreatic lymph nodes was detected by the presence of a focal dark spot with T2* effect in a caudal area of the pancreas. Detection of loaded T cells in pancreatic islets was evaluated by comparison of histograms of MR signal intensity generated in whole pancreas in mice injected with loaded and unloaded T cells. Homing of loaded T cells was confirmed at transmission electronic microscopy (TEM). Fifty-six mice underwent all experiments. RESULTS: Focal dark spots with T2* effect were observed at 11 days in all three mice injected with loaded T cells and in none of the three mice injected with unloaded T cells. At 20 days, a more diffuse negative enhancement of the whole pancreas was noticed in one mouse injected with loaded T cells than in three mice injected with unloaded T cells. Presence of loaded T cells was confirmed with TEM. In vitro and in vivo tests confirmed that survival and function were not altered by loading. CONCLUSION: The ability of MR imaging to depict cell homing in living organisms at least 20 days after cell labeling was demonstrated, opening the way of follow-up in autoimmune diseases and cell therapy.     

MR imaging of biodegradable polymeric microparticles: a potential method of monitoring local drug delivery.

Gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) was encapsulated into biodegradable, bioadhesive polymeric microparticles to enable noninvasive monitoring of their local intravesical delivery with MRI. The microparticles were characterized by contrast agent encapsulation and release kinetics, T(1) relaxation rates, and contrast enhancement in vivo. The level of Gd-DTPA loading into microparticles was 14.3 +/- 0.6 mug/mg polymer. The measured T(1) relaxation rates of the microparticles showed a direct dependence on Gd-DPTA content. Both 1.5T and 4.7T MR scanners were used to image murine bladders instilled intravesically with Gd-DTPA-loaded particles in vivo. MR images showed ring-shaped regions of enhancement inscribing the bladder wall, which were attributed to the microparticles that were preferentially adherent to the mucosa lining the urothelium. The images of controls exhibited no such enhancement. The normalized signal intensities measured from post-instillation images were significantly greater (P < 0.05) than those in the pre-instillation images. Contrast enhancement was observed for at least 5 days after the initial instillation, although the enhancement decreased due to microparticle degradation or mucosa renewal. The localized distribution of biodegradable, bioadhesive microparticles encapsulating Gd-DTPA was successfully visualized with MRI in vivo, allowing particle-mediated delivery to be temporally and spatially monitored noninvasively.     

On the use of [18F]DOPA as an imaging biomarker for transplanted islet mass

Aim

Islet transplantation is being developed as a potential cure for patients with type 1 diabetes. There is a need for non-invasive imaging techniques for the quantification of transplanted islets, as current transplantation sites are associated with a substantial loss of islet viability. The dopaminergic metabolic pathway is present in the islets; therefore, we propose Fluorine-18 labeled l-3,4-dihydroxyphenylalanine ([¹⁸F]DOPA) as a biomarker for transplanted islet mass.

Methods

The expression of enzymes involved in the dopaminergic metabolic pathway was investigated in both native and transplanted human islets. The specific uptake of [¹⁸F]DOPA in islets and immortalized beta cells was studied in vitro by selective blocking of dopa decarboxylase (DDC). Initial in vivo PET imaging of viable subcutaneous human islets was performed using [¹⁸F]DOPA.

Results

DDC and vesicular monoamine transporter 2 are co-localized with insulin in the native human pancreas, and the expression is retained after transplantation. Islet uptake of the [¹⁸F]DOPA could be modulated by inhibiting DDC, indicating that the uptake followed the normal dopaminergic metabolic pathway. In vivo imaging revealed [¹⁸F]DOPA uptake at the site of the functional islet graft.

Conclusion

Based on the in vitro and in vivo results presented in this study, we propose to further validate [¹⁸F]DOPA-PET as a sensitive imaging modality for imaging extrahepatically transplanted islets.     

Feasibility of baculovirus-mediated reporter gene delivery for efficient monitoring of islet transplantation in vivo

ObjectiveThe objective of this study was to explore the feasibility of baculovirus vector-mediated sodium iodide symporter (NIS) gene delivery to monitor islet transplantation.MethodsBaculovirus vectors expressing green fluorescent protein (GFP) or NIS (Bac-GFP and Bac-NIS) were established using the Bac-to-Bac baculovirus expression system. The GFP expression of Bac-GFP-infected rat islets was observed in vitro by fluorescence microscopy. Iodine uptake and inhibition of iodine uptake by NaClO₄ in Bac-NIS-infected islets were dynamically monitored in vitro. Bac-GFP- or Bac-NIS-infected islets were implanted into the left axillary cavity of NOD-SCID mice, and fluorescence imaging and ¹²⁵I NanoSPECT/CT imaging were subsequently performed in vivo.ResultsBac-GFP efficiently infected rat islets (over 95% infected at MOI = 40), and the expression of GFP lasted approximately two weeks. NaClO₄ could inhibit iodine uptake by Bac-NIS-infected islets. In vivo imaging revealed that the fluorescence intensity of the transplant sites in Bac-GFP-infected groups was significantly higher than in the non-infected group. Grafts could be clearly observed by ¹²⁵I NanoSPECT/CT imaging for up to 8 h.ConclusionBaculovirus vectors are powerful vehicles for studying rat islets in gene delivery. It is feasible to use a baculovirus vector to delivery an NIS gene for non-invasive monitoring transplanted islets in vivo by the expression of the target gene.     

供体凋亡脾细胞输注诱导大鼠胰岛移植免疫耐受的研究

目的 探讨供体凋亡细胞输注诱导大鼠胰岛移植免疫耐受的可行性.方法 应用链脲佐菌素(STZ)制备SD大鼠糖尿病模型,以直线加速器照射诱导供体Wistar大鼠脾细胞凋亡.将糖尿病SD大鼠分为4组,分别经阴茎背静脉输注Hanks液、供体正常脾细胞、供体凋亡脾细胞、供体坏死脾细胞,7天后于肾包囊进行同种异体胰岛移植,测定血糖变化,观测胰岛移植物存活时间,并通过混合淋巴细胞反应(MLR)观察移植耐受的状态.结果 预输注供体凋亡脾细胞能显著延长同种异体胰岛移植物存活时间(中位存活时间达31天),并使受体鼠对供体鼠的MLR明显减弱.结论 供体凋亡细胞输注能够诱导同种异体大鼠胰岛移植免疫耐受.     

TIPS术中胆汁漏出：刺激平滑肌细胞增生?

目的经颈内静脉肝内门腔静脉分流术（ＴＩＰＳ）中胆道损伤并胆汁漏出，可能是引起ＴＩＰＳ术后支架内平滑肌细胞（ＳＭＣ）增生和狭窄的重要因素。本研究以离体ＳＭＣ培养和ＴＩＰＳ猪模型实验评价胆汁对ＳＭＣ的作用。材料与方法离体ＳＭＣ培养分为三组：Ⅰ组＝１．０％血清＋１．０％胆汁；Ⅱ组＝１０．０％血清＋１．０％胆汁；Ⅲ组＝１０．０％血清。细胞收获点分别为３、１０、１４天。动物实验组共用４５只猪建立ＴＩＰＳ模型，术后处死时间为１０～１６天。结果此前预试验结果：使用２．５％、５．０％、１０．０％胆汁的培养基，ＳＭＣ在３天内全部死亡。离体细胞培养（１．０％胆汁）实验组（Ⅰ、Ⅱ组）中的脱氧核糖核酸（ＤＮＡ）和总蛋白量明显较对照组（Ⅲ组）少（Ｐ＜０．０５），且随着培养时间的延长其差异增加。动物实验组：２８．８９％的标本证实有胆汁漏出。定量分析示胆汁漏组中的ＳＭＣ增殖量较无胆汁漏组少。组织学检查发现有新生胆管在支架内生长。结论２．５％～１０．０％胆汁杀死ＳＭＣ，１．０％胆汁可抑制ＳＭＣ生长。动物实验示胆汁漏出导致ＳＭＣ增生减少，但血栓形成增加，从而使支架闭塞率增高。     

Evaluation of Porcine Pancreatic Islets Transplanted in the Kidney Capsules of Diabetic Mice Using a Clinically Approved Superparamagnetic Iron Oxide (SPIO) and a 1.5T MR Scanner

Objective

To evaluate transplanted porcine pancreatic islets in the kidney capsules of diabetic mice using a clinically approved superparamagnetic iron oxide (SPIO) and a 1.5T MR scanner.

Materials and Methods

Various numbers of porcine pancreatic islets labeled with Resovist, a carboxydextran-coated SPIO, were transplanted into the kidney capsules of normal mice and imaged with a 3D FIESTA sequence using a 1.5T clinical MR scanner. Labeled (n = 3) and unlabeled (n = 2) islets were transplanted into the kidney capsules of streptozotocin-induced diabetic mice. Blood glucose levels and MR signal intensities were monitored for 30 days post-transplantation.

Results

There were no significant differences in viability or insulin secretion between labeled and unlabeled islets. A strong correlation (r² > 0.94) was evident between the number of transplanted islets and T₂ relaxation times quantified by MRI. Transplantation with labeled or unlabeled islets helped restore normal sustained glucose levels in diabetic mice, and nephrectomies induced the recurrence of diabetes. The MR signal intensity of labeled pancreatic islets decreased by 80% over 30 days.

Conclusion

The transplantation of SPIO-labeled porcine islets into the kidney capsule of diabetic mice allows to restore normal glucose levels, and these islets can be visualized and quantified using a 1.5T clinical MR scanner.